Intestinal Microbial Metagenome Sequencing And Microorganisms Culture

1.In this thesis, a method for evaluating the genomic DNA extracted from shrimp intestinal microfloar based on semi-quantitative PCR was developed. Method: Three approaches were applied in extracting intestinal metagenomic DNAs in L.vannamei:1) TIANamp bacteria DNA kit,modified CTAB- phenol/chloroform method,pre-treated with RNAdungeon reagent. Bacterial 16S rDNA and L.vannamei 18S rDNA were amplified and compared for the ratio of bacterial and host DNA contents. The results suggested that the concentration of metagenome were 92.5ng/ L,24.8ng/ L and microbial metagenome ratio were 72.3%, 67.6%, respectively. We have tested RNAdugeon for sample storage in metagenomic research. The DNA concentration of sample pre-treated by RNAdungeon was 36.7ng/ L and microbial metagenome ratio was up to 89.9%. Solexa sequencing indicated that up 64.1% of congtigs in metagenome has no assignment or no hits in database NCBI, where 35.5% contiges was eukaryote, and low 0.4% sequences belongs to bacteria or virus.2. In this thesis, different bacterial culture methods were studied for shrimp symbionts. By using 2216E medium for intestinal bacteria culture of Litopenaeus vannamei and by using the detection of different bacteria growth was based on PCR-RFLP (restriction Fragment Length Polymorphism) analysis of 16S rDNA gene. Our results suggest there are up to 28 different bacteria 16S rDNA RFLP patterns in control group where liquid\solid \ semisolid medium only have14, 8 and 6, respectively. Among those RFLP patterns, two were found in all groups and 6 RFLP have no distribution in liquid\solid \ semisolid medium groups. And the first major RFLP pattern in control group which is up to 49% was no distribution in culture groups. Conclusion: different physical property of medium has influence on bacteria growth and the major bacteria species in intestine of Litopenaeus vannamei was non-cultured through 2216E medium. Some bacteria cannot be shown as colony forming units by the conventional plate counts were not unculturable but non-cultured for they may have growth in liquid medium.